Quantification of bacterial chemotaxis by measurement of model parameters using the capillary assay

The capillary assay for quantitative characterization of bacterial motility and chemotaxis is analyzed in terms of a mathematical model for cell population migration, in order to determine values for the cell random motility coefficient, mu and the cell chemotaxis coefficient, chi. The analysis involves both analytical perturbation methods and numerical finite-difference techniques. Transient cell density profiles within the capillary tube are determined as they depend upon mu and chi, providing a means for estimating mu and chi from the common protocol measurements of cell accumulation in the tube at specified observation times. The effects of extraneous factors such as assay geometry, stimulus diffusivity, bacterial density, and observation time are thus separated from the intrinsic cell-stimulus interaction and response. This allows independent population measurements of cell chemosensory movement properties to be extrapolated to situations involving growth and competition of populations, for purposes of better understanding microbial population dynamics in systems of biotechnological and microbial ecological importance.     

Water transport by the bacterial channel alpha-hemolysin

This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels.     

Selectivity enhancement of an Escherichia coli bacterial electrode using enzyme and transport inhibitors

The feasibility of using specific enzyme and transport inhibitors to minimize the glutamine response of a potentiometric microbial sensor is demonstrated. The glutamine response of a bacterial electrode prepared with Escherichia coli as the biocatalyst in conjunction with an ammonia gas-sensing electrode was greatly reduced by treating the electrode with the enzyme inhibitor 6-diazo-5-oxo-L-norleucine (DONL) and the transport inhibitor gamma-L-glutamylhydrazide. Each inhibitor effectively decreased glutamine response to a level sufficiently low to be considered negligible in clinical studies. Although the sensor ultimately recovered from the effects of a single exposure to an inhibitor, continuous exposure at an optimum concentration maintained a low response to glutamine. Furthermore, the treatment of the sensor with both inhibitors simultaneously resulted in a negligible response to glutamine of <1 mV, indicating that both inhibitors are necessary for optimum inhibition of glutamine response. This approach is promising as a means of enhancing the selectivity of microbial sensors.     

Protein transport by the bacterial Tat pathway

The twin-arginine translocation (Tat) system accomplishes the remarkable feat of translocating large – even dimeric – proteins across tightly sealed energy-transducing membranes. All of the available evidence indicates that it is unique in terms of both structure and mechanism; however its very nature has hindered efforts to probe the core translocation events. At the heart of the problem is the fact that two large sub-complexes are believed to coalesce to form the active translocon, and ‘capturing’ this translocation event has been too difficult. Nevertheless, studies on the individual components have come a long way in recent years, and structural studies have reached the point where educated guesses can be made concerning the most interesting aspects of Tat. In this article we review these studies and the emerging ideas in this field. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.     

Optimisation of polymeric surface pre-treatment to prevent bacterial biofilm formation for use in microfluidics

The production of a microfluidic device for microbial culture has necessitated the development of techniques for the prevention of bacterial adhesion to a range of polymeric substrates including fluoropolymers such as fluorinated ethylene polypropylene, and polyolefins such as low-density polyethylene. Treatment of such materials to increase hydrophilicity reduces the incidence of attachment of Escherichia coli during the first 4 h of cultivation, although no decrease in the number of biofilm initiation sites was detected after 16 h. The incorporation of a mannose analogue to block binding proteins on the F1 binding fimbriae was also investigated. The possibility of ensuring suspension culture of bacterial cells in high surface area to volume ratio nano-vessels is thus facilitated by the correct choice and pre-treatment of materials used in their construction.     

Experimental verification of Marr-Albus' plasticity assumption for the cerebellum

Marr-Albus model of the cerebellum is based on the assumption that parallel fiber-Purkinje cell synapses are plastically modifiable under influences of conjunctive activation of a climbing fiber and a mossy fiber on the same Purkinje cell. This article introduces a positive support for this assumption derived from the recent experiment by Ito. Sakurai and Tongroach on rabbit flocculus. Possible reasons why the experimental verification of Marr-Albus' assumption has been so difficult are considered, and applicability of Marr-Albus model of the cerebellum to problems of motor learning is discussed.     

Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58ngμL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34ngμL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3)copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2)copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.     

Membrane transport parameters in frog corneal epithelium measured using impedance analysis techniques

Summary Active Cl^– transport in bullfrog corneal epithelium was studied using transepithelial impendance analysis methods, and direct-current (DC) measurements of membrane voltages and resistance ratios. The technique allows the estimation of the apical and basolateral membrane conductances, and the paracellular conductance, and does not rely on the use of membrane conductance-altering agents to obtain these measurements as was requisite in earlier DC equivalent-circuit analysis studies. In addition, the analysis results in estimates of the apical and basolateral membrane capacitances, and allows resolution of the paracellular conductance into properties of the tight junctions and lateral spaces. Membrane capacitances (proportional to areas) were used to estimate the specific conductances of the apical and basolateral membranes, as well as to evaluate coupling between the cell layers. We confirm results obtained from earlier studies: (1) apical membrane conductance is proportional to the rate of active Cl^– transport and is, highly Cl^– selective; (2) intracellular Cl^– activity is above electrochemical equilibrium, thereby providing a net driving force for apical membrane Cl^– exit; (3) the paracellular conductance is comparable to the transcellular conductance. We also found that: (1) the paracellular conductance is composed of the series combination of the junctional conductance and a nonnegligible lateral space resistance; (2) a small K⁺ conductance reported in the apical membrane may result from Cl^– channels possessing a finite permeability to K⁺; (3) the basolateral membrane areas is 36 times greater than the apical membrane area which is consistent with the notion of electrical coupling between the five to six cell layers of the epithelium; (4) the specific conductance of the basolateral membrane is many times lower than that of the apical membrane; (5) the net transport of Cl^– is modulated primarily by changes in the conductance of the apical membrane and not by changes in the net electrochemical gradient resulting from opposite changes in the electrical and chemical gradients; (6) the conductance of the basolateral membrane does not change with transport which implies that the net driving force for K⁺ exit increases with transport, possibly due to an increase in the intracellular K⁺ activity.     

Experimental verification of the 'stability profile of mutant protein' (SPMP) data using mutant human lysozymes.

The stability profile of mutant protein (SPMP) (Ota,M., Kanaya,S. and Nishikawa,K., 1995, J. Mol. Biol., 248, 733-738) estimates the changes in conformational stability due to single amino acid substitutions using a pseudo-energy potential developed for evaluating structure-sequence compatibility in the structure prediction method, the 3D-1D compatibility evaluation. Nine mutant human lysozymes expected to significantly increase in stability from SPMP were constructed, in order to experimentally verify the reliability of SPMP. The thermodynamic parameters for denaturation and crystal structures of these mutant proteins were determined. One mutant protein was stabilized as expected, compared with the wild-type protein. However, the others were not stabilized even though the structural changes were subtle, indicating that SPMP overestimates the increase in stability or underestimates negative effects due to substitution. The stability changes in the other mutant human lysozymes previously reported were also analyzed by SPMP. The correlation of the stability changes between the experiment and prediction depended on the types of substitution: there were some correlations for proline mutants and cavity-creating mutants, but no correlation for mutants related to side-chain hydrogen bonds. The present results may indicate some additional factors that should be considered in the calculation of SPMP, suggesting that SPMP can be refined further.     

Quantification of random motility and chemotaxis bacterial transport coefficients using individual-cell and population-scale assays

A number of individual-cell and population-scale assays have been introduced to quantify bacterial motility and chemotaxis. The transport coefficients reported in the literature, however, span several orders of magnitude, making it difficult to ascertain to what degree variations in bacterial species/strain, growth medium, growth and experimental conditions, and experiment type contribute to the reported differences in coefficient values. We quantified the random motility of Escherichia coli AW405 using the capillary assay, stopped-flow diffusion chamber (SFDC), and tracking microscope. We obtained good agreement for the random motility coefficient between these assays when using the same bacterial strain and consistent growth and experimental conditions. Chemotaxis of E. coli toward the attractant alpha-methylaspartate was quantified using the SFDC and capillary assay. Good agreement for the chemotactic sensitivity coefficient between the SFDC and the capillary assay was obtained across a limited attractant concentration range. Three different mathematical models were considered for analyzing capillary assay data to obtain a chemotactic sensitivity coefficient. These models differed by their treatment of the bacterial concentration in the chamber and the attractant concentration at the mouth. Results from our study indicate that the capillary assay, the most commonly used bacterial random motility and chemotaxis assay, can be used to accurately quantify bacterial transport coefficients over a limited range of attractant concentrations, provided experiments are performed carefully and appropriate mathematical models are used to interpret the experimental data.     

Experimental verification of the method for detection of water microleakages in plasma vacuum chambers by using the hydroxyl spectrum

Experimental determination of the sensitivity of the method for detection of water microleakages in the cooling systems of the plasma vacuum chambers of complex electrophysical devices (such as tokamaks, fuel elements of nuclear reactors, and plasmachemical reactors) is considered. It was shown that the spectroscopic method for detection of water microleakages by using the hydroxyl radiation spectrum makes it possible to detect leakages at a level of 10⁻⁵ Pa m³ s⁻¹. The spatial resolution of the method allows one to localize defects with an accuracy of several centimeters.     

Experimental verification of the opponent theory of human color vision

The colors observed by the human eye after a short flash of light of different spectral compositions were studied experimentally. The successive images and changes in their color with time confirm the opponent theory of human color vision.     

The enzymology of the bacterial phosphoenolpyruvate-dependent sugar transport systems

Summary The phosphoenolpyruvate-dependent sugar transport system (PTS) is present in a large variety of bacteria. It catalyzes transport and phosphorylation of hexoses and hexitols at the expense of phosphoenolpyruvate. Only three of four enzymes are required for this entire sequence. Each component has been isolated and purified to the homogeneity from one bacterial species or another allowing recent investigations intomechanistic aspects of energy coupling, energy conservation, transport and regulation using well-characterized enzymes. In each case the phosphorylation of the enzyme is a key element in that enzymes function.The initial step in the energy conversion process is the E_I catalyzed conversion of phosphoenolpyruvate to pyruvate and P-HPr. E_II is a metal requiring hydrophobic enzyme which is active only as a dimer. Kinetic and gel filtration data confirm that it forms functional ternary complexes with HPr or P-Hpr and phosphoenolpyruvate or pyruvate which influence both the degree of dimerization and the specific activity of the dimer. The dimer appears to carry only one phosphoryl group suggesting that negative cooperativity or a flip-flop mechanism may be involved in the sequence of phosphoryl group transfer.Many of the PTS phosphoenzyme intermediates carry the phosphoryl group as a phospho-histidine. A general mechanism for the transfer of the phosphoryl group to and from the active site histidine residue in each protein has been established with high resolution ¹H NMR data. At physiological pH the active site histidine is deprotonated, whereas the phosphohistidine is protonated. Consequently the histidine, as a strong nucleophile, can abstract the phosphoryl group from the donor while protonation destabilizes the phosphohistidine facilitating passage of the phosphoryl group to the following enzyme intermediate. The change in protonation state accompanies a phosphorylation induced conformational change in the carrier.The ability of the PTS to regulate the activity of other permeases and catabolic enzymes has been attributed to E_III ^Glc. Data obtained with mutants suggest that changes in the phosphorylation state alter the regulatory properties of the enzyme. The nonphosphorylated species blocks various permeases and suppresses adenylate cyclase activity thereby inhibiting the synthesis of catabolic enzyme systems. The phosphorylated species stimulates adenylate cyclase and permits the uptake of inducers leading to the initiation of catabolic enzyme synthesis. Experiments with the isolated E_III ^Glc confirm that a phosphoenzyme intermediate exists.Transport and phosphorylation of the sugar are catalyzed by a membrane-bound E_II via a phosphoenzyme intermediate which can be reached from P-HPr, P-E_III or sugar-P. The phosphorylation state controls the affinity of the enzyme for its substrates. E_II is high affinity for P-HPr or P-E_III and low affinity for sugar. P-E_II is high affinity for sugar and low affinity for P-HPr or P-E_III. The affinity of the enzyme for sugar substrates is controlled by the oxidation state of a dithiol. The reduced, dithiol form is high affinity for sugar substrates. The oxidized, disulfide form, is low affinity. Phosphorylation of the enzyme chould shift the affinity for substrates by altering the oxidation state of the enzyme.     

Experimental verification of the model of complement-dependent immune lysis of liposomes

The quantitative dependences of the complement-dependent immune lysis of a monodisperse suspension of 200-nm liposomes sensitized by a monovalent hapten (2,4-DNP-ɛ-caproyl-DPPE) or a polyvalent antigen (LPS from Francisella tularensis) on the initial concentration of specific antibodies (IgG) to the hapten and antigen have been investigated. The quantity of antibody-binding sites on the liposome surface was evaluated. The difference between the complement-dependent lysis of poly- and monodisperse suspensions of liposomes was shown. The experimental results are well described by the direct binding model.